The present invention relates to a cell culture technique. More specifically, it relates to a carrier for cell culture, to a method for culturing cells using the carrier, to a cultured cell layer obtained by this method, to a method for piling up another cell layer on the cultured cell layer, and a cell multi-layer obtained by this piling up method.
Since hydrated polymer gel has a bio-mimetic structure and has expanding or contracting properties depending on external conditions such as temperature or acidic/alkaline condition, it has been attempted not only to apply such a gel to an artificial organ or tissue such as artificial muscle, and to use it in the medical field for controlling the amount of release of a drug which has been encapsulated within the gel, but also to use it as a support for cell growth being the gel which comprises various types of cytokines or the like.
Among hydrated polymer gels, inter alia N-isopropylacrylamide (referred to as xe2x80x9cNIPAMxe2x80x9d hereafter), which is a temperature responsive polymer, can swell and is in a liquid state at a low temperature; however, it occurs phase transition at around 34xc2x0 C. thereby resulting in rapid contraction and gelation.
So far, for the purpose of stratification of cultured cell layers, a method has been used, wherein the cell cultured on NIPAM gel was piled up on another cell layer together with the NIPAM at a temperature of 37xc2x0 C., subsequently the NIPAM was liquefied by lowering the temperature below 34xc2x0 C. to be removed, whereby those cells were directly piled up each other.
Generally, when cell is cultured on NIPAM, it grows in the form of a monolayer, forming Extracellular Matrix (referred to as xe2x80x9cECMxe2x80x9d hereafter) such as collagen between two adjacent cells. In this case the cell must attach to ECM for its growth.
However, because the upper side of cell layer, as well as the region between the cell and NIPAM (as a basal layer), is not attached to another cell, ECM, which is necessary for cell adhesion, is not formed.
Thus, even if monolayers of cells cultured on NIPAM are piled up each other, and then the NIPAM is removed by solubilizing under temperature conditions below 34xc2x0 C. to pile up the cell layers so as to be contacted directly each other, the support is not enough for the cell overlaid above to be proliferated, accordingly, stable proliferation could not be expected.
Since the phenomenon is seen that the liquefied NIPAM also acts as cytotoxin to inhibit the normal cell growth, the above method was a very unsuitable and unstable technique as a means of cell stratification.
So far, there was no successful example of culturing a cell on a medium prepared by piling up the ECM component on various types of gels followed by gelation, and establishing a culture system has been attempted, the system employing a medium wherein the gel, no longer required after piling up, can be readily removed.
In those circumstances, the object of the present invention is to provide a technique for piling up cells in a stable and easy manner without using NIPAM which inhibits the cell growth and proliferation when it is solubilized, and a technique wherein cells on upper and lower sides can be adhered to each other via ECM (e.g. collagen) when the cell layers are piled up; that is, the invention is aimed at establishing a technique for the cell multi-layer which has been considered to be difficult to achieve in vitro except certain tissues including skin.
Alginic acid is a block copolymer composed of glucuronic acid (G) and mannuronic acid (M), wherein glucuronic acid forms an egg-box structure such that it surrounds a multivalent metal ion (e.g. calcium ion), thereby forming an alginate gel (see FIG. 1). The greater the G/M ratio is, the higher the ability of alginic acid to form the gel becomes. However, since the alginate gel can not be molten even at a temperature above 100xc2x0 C., the method using a temperature sensitive substance like NIPAM is rather inappropriate as a method for exfoliating a living cell from alginate gel. On the other hand, the alginate gel is easily dissolved and liquefied when being soaked in a chelating agent (e.g. EDTA). Additionally, since the alginate gel is a natural product belonging to algae with a property of biodegradability, it does not inhibit the normal cell growth even when the alginate gel is not removed sufficiently upon its dissolution.
As a result of the extensive and intensive studies that were focused on the above properties of alginate gel, the present inventors have now found that it is easy to pile up a cell layer on an another cell layer when a cell is cultured using a carrier which comprises an alginate gel layer (e.g. calcium alginate gel layer) piled up on a porous membrane, followed by solubilizing the alginate gel layer. In this method utilizing the alginate gel, the cell culture containing cell layers can readily be detached.
The present inventors have also found that depending on types of cells to be cultured with the carrier for cell culture, the culture of cells can be carried out more efficiently by using a carrier for cell culture wherein ECM component gel layer (e.g. collagen gel layer) or ECM component sponge layer (e.g. collagen sponge layer) is further piled up on the alginate gel layer which has been piled up on a porous membrane.
The present invention was accomplished on the basis of the above described findings.
Thus, the present invention includes the following inventions:
(1) A carrier for cell culture comprising a porous membrane and an alginate gel layer which is formed on the membrane.
(2) The carrier of (1), wherein the alginate gel layer is composed of a calcium alginate gel.
(3) The carrier of (1) or (2), which further comprises an extracellular matrix component gel layer or extracellular matrix component sponge layer which is formed on the alginate gel layer.
(4) The carrier of (3), wherein the extracellular matrix component is collagen.
(5) A method for culturing a cell, wherein the method comprises culturing the cell using the carrier for cell culture according to any one of (1) to (4).
(6) A method for piling up a cell, wherein the method comprises: forming a cell layer on the carrier according to any one of (1) to (4); solubilizing an alginate gel layer of the carrier thereby exfoliating the cell layer from a porous membrane of the carrier; and piling up the exfoliated cell layer on another cell layer formed on the carrier according to any one of (1) to (4).
(7) A cell multi-layer obtained by the method according to (6).